1. Field of the Invention
The present invention relates generally to viral vectors and their use as expression vectors for transforming human cells, both in vitro and in vivo. More specifically, the invention relates to adenoviral expression constructs comprising a proapoptotic member of the Bcl-2 gene family.
2. Description of Related Art
Adenoviral vectors have become one of the leading vectors for gene transfer, particularly in gene therapy contexts. These vectors have been studied rigorously in both in vitro and in vivo contexts because of the ability to generate high titer stocks, their high transduction efficiency and their ability to infect a variety of tissue types in different species. In addition, the availability of cell lines to complement defects in adenoviral replication functions provides for the use of replication defective mutants carrying, in the place of selected structural genes, recombinant inserts of interest.
Several studies have demonstrated the ability of adenovirus-mediated wild-type p53 replacement gene therapy to induce a G1 cell cycle arrest and/or apoptosis in malignant cells carrying p53 gene mutations. Though the mechanism of G1 arrest via p21 and the cyclin-dependent kinase pathway has been widely studied, little is known of the mechanisms by which wild-type p53 induces apoptosis. It appears that p53 induces apoptosis, at least in part, by up-regulating proapoptotic members of the Bcl-2 family of proteins.
The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. Apoptosis, or programmed cell death, is an essential occurring process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins which can be categorized as death agonists or death antagonists.
Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli which will be discussed in detail. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 or counteract Bcl-2 function and promote cell death.
One such family member having Bcl-2 counteracting function is Bax. Bax, Bcl-2 associated X protein, is a death agonist member of the Bcl-2 family of proteins (Oltvai et al., 1993). It has been suggested that Bax may function as a primary response gene in a p53 regulated apoptotic pathway (Miyashita et al., 1994). Indeed, it has been shown that there is a p53 consensus binding region in the promoter region of the proapoptotic Bax gene (1995). Bax mRNA and protein expression are increased following induction of p53. However, the observed induction of p53-dependent apoptosis in Bax knock out mice clearly indicates that other pathways or proteins are involved. Bak, a Bcl-2 homologue, is expressed in a variety of tissues and has been demonstrated to induce program cell death independent of Bax expression (Krajewski et al., 1996; Chittenden et al., 1995). The accumulation of Bak protein in cells infected with Adp53, may be an additional mechanism by which p53 can induce programmed cell death.
However, a recent report has demonstrated an increase in Bcl-xL, expression following wild-type p53 expression in the human colorectal cancer cell line HT29 (Merchant et al., 1996). The authors hypothesize that this increase expression may lead to an inhibition of program cell death pathways and accounted for lack of p53-induced apoptosis observed in these cells. Another potential problem with p53 therapy is that the amount of viral material administered provides risks of host cell toxicity and/or immune response. Thus, any method which would increase the effect of p53 at low doses, or circumvent the need for high viral doses, would be advantageous.
Given that p53 gene therapy is a powerful tool in the fight against cancer, therapeutic compositions that may augment or complement p53 will serve to improve the currently available cancer therapy regimens. Indeed, compositions that provide the apoptotic effect of p53 without the need for p53 itself would be additionally useful.